Electrostatic Ratchet for Successive Peptide Synthesis in Nonribosomal Molecular Machine RimK.

A nonribosomal peptide-synthesizing molecular machine, RimK, adds l-glutamic acids to the C-terminus of ribosomal protein S6 (RpsF) in vivo and synthesizes poly-α-glutamates in vitro. However, the mechanism of the successive glutamate addition, which is fueled by ATP, remains unclear. Here, we investigate the successive peptide-synthesizing mechanism of RimK via the molecular dynamics (MD) simulation of glutamate binding. We first show that RimK adopts three stable structural states with respect to the ATP-binding loop and the triphosphate chain of the bound ATP. We then show that a glutamate in solution preferentially binds to a positively charged belt-like region of RimK and the bound glutamate exhibits Brownian motion along the belt. The binding-energy landscape shows that the open-to-closed transition of the ATP-binding loop and the bent-to-straight transition of the triphosphate chain of ATP can function as an electrostatic ratchet that guides the bound glutamate to the active site. We then show the binding site of the second glutamate, which allows us to infer the ligation mechanism. Consistent with MD results, the crystal structure of RimK we obtained in the presence of RpsF presents an electron density that is presumed to correspond to the C-terminus of RpsF. We finally propose a mechanism for the successive peptide synthesis by RimK and discuss its similarity to other molecular machines.

Structural polymorphism of the Escherichia coli poly-α-L-glutamate synthetase RimK.

Bacterial RimK is an enzyme that catalyzes the polyglutamylation of the C-terminus of ribosomal protein S6 and the synthesis of poly-α-L-glutamate peptides using L-glutamic acid. In the present study, the crystal structure of the Escherichia coli RimK protein complexed with the ATP analogue AMP-PNP was determined at 2.05 Šresolution. Two different conformations of RimK, closed and open forms, were observed in the crystals. The structural polymorphism revealed in this study provided important information to understand the mechanism by which RimK catalyzes the synthesis of poly-α-L-glutamate peptides and the polyglutamylation of ribosomal protein S6.